Laboratory-scale culture and testing of cells and tissues derived from mammalian sources typically involves the use of specialized containers. The physical and chemical nature of the chosen vessel dictates the choice of handling methods and technical limitations of the experimental process. Static vessels such as petri dishes and tissue flasks, roller bottles which are rotated to provide continuous bathing of cells which grow attached to the walls of the vessel, or spinner flasks in which a moving paddle continually suspends cellular material in nutrient broth or media are commonly employed. In all of these approaches, the chosen vessel must be associated with a laminar flow hood for aseptic set-up and servicing. Furthermore, these vessels do not have independent means for controlling temperature. As such, such vessels must be placed within an incubator designed to regulate temperature and control atmosphere during maintenance.
In a further limitation, such vessels are, by design, open systems having direct gaseous communication with the ambient environment. For this reason, accidental contamination of the contents of the vessel with atmospherically borne microbial elements is common. When contamination with such ambient elements occurs, the user must reject the contents of the container from the particular study at hand, thereby causing loss of data and time. Systems which employ vessels, such as those listed above, are highly labor intensive, inconvenient, expensive, unreliable for maintenance of sterility and may also be wasteful of laboratory space.
While the prior art does evidence the existence of devices which, in limited ways, may be employed to grow and maintain living cells and tissues, most of these devices are incorporated into bioreactor-type inventions which utilize cells as living chemical fabricators to produce proteins, enzymes, monoclonal antibodies, hormones, drugs, pesticides and other substances. In such devices, the focus is on the desired end product and not on the cells themselves. The cells are simply the means to the end product meant for applications outside the system.
Given the limitations of known systems Concerning sterility, contamination, reproducibility of results, size, expense and reliability, a need has developed for a single self-contained device able to sustain life of living cells and tissues, grow the tissues, facilitate performance of experimentation on the tissues and obtain results of such experimentation, free of risk of contamination, loss of sterility, and in a reproducible manner. It is with these thoughts in mind that the present invention was developed.
U.S. Pat. No. 4,725,548 to Karrer discloses a method and fermenter for growing tissue cells. In the Karrer device, once the cells are grown, they are transferred from the device via a harvest pipe for use external to the Karrer device. The present invention differs from the teachings of Karrer as contemplating a completely self-contained device wherein cells and other living tissues may be grown and wherein testing and experimental procedures utilizing these living tissues and cells occur within the confines of the completely self-contained device.
Several devices and systems are known in which cells and tissues are grown in gas permeable plastic bags. Those patents known to Applicant which employ such structure are U.S. Pat. Nos. 3,102,082 to Brewer, 3,941,662 to Munder et al., 4,142,940 to Modolell et al., and 4,829,002 to Pattillo et al. In such devices, the containers may be filled aseptically with media and bulk additives. System implementation is hampered by inherently limited cellular capacity and tedious turnover of media following application of hormones or drugs to the cultures. Systems such as those disclosed in these patents require use of an incubator to provide thermal and atmospheric maintenance. Passaging of cultured cells at confluence is done manually and the cells must be removed physically from the system. This transfer technique may facilitate contamination of cultured cells by microorganisms. Furthermore, the systems disclosed in these patents do not permit monitoring of the metabolic status of cells during culture. The present invention overcomes these deficiencies in these prior art designs.
An alternative to the static bag culture system is the hollow fiber bioreactor approach described in the Knazek et al. patents, U.S. Pat. Nos. 3,821,087, 3,883,393, 4,184,922, 4,200,689, 4,206,015 and 4,220,725. These systems employ membrane capillaries encased in rigid plastic housings sealed in such a manner that two discrete volumes are established which communicate via pores of molecular dimensions which traverse the membranes. The cells are inoculated into a static volume external to the capillaries. A mobile volume is contained within the membrane proper and the connected vessels and conduits. This volume may include a replenishable media reservoir and gas permeable tubing or membrane oxygenating devices which serve to adjust acidity in the media by allowing equilibration with controlled concentrations of carbon dioxide and facilitating uptake of oxygen. The media are circulated by means of a pump. While very high cell density is obtainable with the systems disclosed in these patents, direct observation of cells within the system is not feasible. In a further aspect, set-up must be accomplished under a laminar flow hood for aseptic set-up and servicing. Additionally, the systems disclosed in these patents must reside within a carbon dioxide incubator or equivalent cabinet. Furthermore, application of hormones or drugs is difficult and passaging of cells is manual, leading to potential for biological contamination.
U.S. Pat. No. 4,650,766 to Harm et al. discloses a culturing apparatus which is compatible with hollow fiber bioreactors such as those disclosed in the Knazek et al. patents. Patentees Harm and Peluso describe a culture apparatus which provides integral gassing of media and provision of heating to eliminate the need for incubators. The patented apparatus is limited by the fact that the system is of a single pass design resulting in inefficient use of media. Fluid flow is very slow resulting in loss of water through the exchange membrane. The osmolarity of the media increases to the detriment of the cells or tissues under culture. Dissolved gas components are the limiting nutrients in the system and new media is constantly moved over the cells to provide these materials. Open lines run from the outlet of the bioreactors to a supplemental fraction collector. The termination of these lines is open to the environment providing ready access for microbial contamination of the system. Gassing and heating of the medium is concurrent favoring formation of gas bubbles in the flow path. As is well known, presence of bubbles in a flowing system is lethal to cells in culture. Multiple media sources are mixed and distributed via manifolds thereby providing the potential for a single contamination nidus to infect all bioreactors in the system. While parallel flow paths are possible in the design, to thereby provide potential for reduction in the impact of casual contamination, gassing is continuous resulting in waste of supplies. Thermal regulation is determined by external water bath controls allowing the potential for rapid temperature variations which may be lethal. The Harm et al. system provides no automated control or monitoring nor is any facility for the introduction of drugs or hormones provided. Additionally, no computer control is provided.
U.S. Pat. No. 4,629,686 to Gruenberg discloses an apparatus for delivering a controlled dosage of a chemical substance to cell or tissue cultures. In the Gruenberg system, a series of pre-diluted media preparations are selectively applied to the organ or tissue of interest. A computer controls the selection of which concentration to apply. The system is single pass in design but does include a sterile dispenser for elimination of potential contamination via the fraction collector/emitter. Temperature is maintained via a circulating water bath providing potential for rapid temperature fluctuations which may be lethal to the tissues contained therein. The Gruenberg device may not be utilized for large scale testing of multiple drugs. The present invention differs from the teachings of Gruenberg as providing a multiple pass system, at least two levels of containment, provision for testing of multiple samples with multiple drugs simultaneously and as including extremely close control over nutrient supply, oxygenation and temperature.
U.S. Pat. No. 4,116,778 to Belousov et al. discloses a plant for continuous cultivation of microorganisms. Belousov et al. fail to contemplate administration of drugs or other substances to living tissues nor monitoring of results of such administration. Furthermore, Belousov et al. provide no control of the temperature of the cells therein. Other significant differences from the present invention also exist.
U.S. Pat. No. 4,894,342 to Guinn et al. discloses a bioreactor based fermenting device designed to grow cell products. Guinn et al. include a humidification process for gas, bubble arrestors and optical fluid level monitoring as well as color detectors. Temperature control is provided through flowing fluid leading to the potential for rapid temperature variations which would be lethal to living tissues. Furthermore, the Guinn et al. system offers no protection to the operator from accidental exposure to infectious or toxic materials.
U.S. Pat. No. 4,446,229 to Indech discloses a method of tissue growth wherein fetuses are transplanted to a unit which circulates blood through an artificial vasculature lung and kidney apparatus. The unit is subjected to ultraviolet light from an integral source to provide sterilization. Such devices usually generate ozone which is toxic to the living tissues. However, Indech fails to disclose any provision for removal of ozone which is generated. A base tissue of non-immunoreactive mesentery is presented to the transplant to which an outgrowth of vasculature is encouraged. Indech claims a method for aseptic addition of materials into the system for testing purposes with respect to the implanted tissue or fetus. Indech fails to disclose methods employed to maintain a sterile barrier. The Indech device is only applicable concerning tissues, embryos and fetuses which are competent to form vasculature tissue denovo.
U.S. Pat. No. 4,889,691 to Argentieri discloses a modular tissue superfusion chamber including a receptacle support having a recess for receiving one of a plurality of modular bath containers which hold tissue samples being tested for electrophysiological responses to commands. While the receptacle support includes a Peltier heater therein, the tissues are exposed to the environment and, as such, may not be isolated from potential contamination.
U.S. Pat. No. 4,680,266 to Tschopp et al. discloses a cell culture chamber with means for automatic replenishment of nutrient. Tschopp et al. disclose a device which may be utilized for carrying out or implementing biological experiments under zero gravity conditions. While this aspect is in common with the teachings of the present invention, the present invention differs from the teachings of Tschopp et al. in many respects. Firstly, the Tschopp et al. device is non-mechanical in nature and relies on osmotically pumped fluid moving through channels drilled into the body of the apparatus, passing over attached cells which reside in a cavity and grow on removable glass windows. The spent media is conveyed into the general cavity of the apparatus. The unit is not self-sustaining and must reside in an incubator so that thermal and atmospheric conditions may be regulated. The device of Tschopp et al. may not be replenished or serviced and cannot be sampled during the course of an experiment.
U.S. Pat. No. 3,065,148 to Ferrari, Jr. discloses a method and apparatus for use in conducting studies on cells. The Ferrari, Jr. device uses very short-term exposure of the cells to test materials and relies upon changes in the rate of carbon dioxide evolution by the cells as an indicator. The individual cells may be recovered in this test scheme and effluent may be fractionated to test for the release of recognized indicators of cell stress and toxic response. Since most cells in the mammalian body are anchorage-dependent, this testing scheme is of severely limited utility in current state of the art laboratories. The Ferrari, Jr. device may not be effectively used for toxicity and carcinogenicity screening.